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Antioxidants reduce oxidative stress in follicular fluid of aged women undergoing IVF

View Article: PubMed Central - PubMed

ABSTRACT

Background: The status characterized by the imbalance between pro-oxidants and antioxidants molecules, defined as oxidative stress, has been suggested to be involved in the pathogenesis of subfertility in females. This study aims to evaluate the impact of a complete micronutrients supplementation on oxidative stress levels in follicular microenvironment as well as on in vitro fertilization (IVF) outcome.

Methods: This preliminary study was conducted between January 2014 and July 2015 at the Siena University Hospital Infertility Clinic. Serum and follicular fluid were collected from infertile women aged > 39 years who underwent two in vitro fertilization cycles: in the first cycle they were treated with GnRH-antagonist protocol and gonadotropins for controlled ovarian hyperstimulation, whereas in the second cycle ovarian stimulation protocol was associated to micronutrients supplementation, starting three months earlier. Protein oxidation levels and total antioxidant capacity in serum and in follicular fluid were evaluated in IVF cycles with or without micronutrients supplementation. Differences in IVF outcome parameters were statistically evaluated.

Results: Two-dimensional electrophoresis analyses demonstrated that when patients assumed micronutrients before IVF cycles, follicular fluid and serum proteins were protected from oxidative damage. Comparable results were obtained when total antioxidant capacity was measured. Moreover, the mean number of good quality oocytes retrieved when patients received micronutrients supplementation was significantly increased.

Conclusion: The additional treatment with micronutrients, starting three months before IVF cycles, protects the follicular microenvironment from oxidative stress, thus increasing the number of good quality oocytes recovered at the pick up.

No MeSH data available.


Avidin-blotting after two dimensional electrophoresis of biotin-labeled free-SH residues in serum (a, b) and follicular fluid (c, d) from untreated patient (a, c) or patient treated with micronutrients supplementation (b, d). The values are the mean ± SD. Statistically significant difference in relative intensity is indicated (*P < 0.05)
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Fig2: Avidin-blotting after two dimensional electrophoresis of biotin-labeled free-SH residues in serum (a, b) and follicular fluid (c, d) from untreated patient (a, c) or patient treated with micronutrients supplementation (b, d). The values are the mean ± SD. Statistically significant difference in relative intensity is indicated (*P < 0.05)

Mentions: Demographic features and clinical data of patients are presented in Table 1. Total FSH-LH units, number of days of stimulation, peak E2 levels at hCG administration were not significantly different between the two IVF cycles (without or with micronutrients supplementation) in the enrolled patients. In order to detect the oxidative stress levels, we measured the total antioxidant capacity and ROS induced-damage on proteins both in follicular fluid and in serum. By means of the FRAP assay we measured total antioxidant capacity showing a significant increase in TAC both in follicular fluid and in serum of women treated with micronutrient supplementation (Fig. 1). In order to confirm these data, we analyzed all samples by two dimensional electrophoresis to monitor the presence of free-SH residues in serum and follicular fluid proteins. In Fig. 2, a representative 2D-electroforesis of serum and follicular fluid proteins is showed. By this procedure, numerous spots distributed in a pH range of about 4–7 with an apparent molecular weight ranging between 15 and 75 kDa (Fig. 2a and c) were evident in patients not receiving micronutrient supplementation (untreated) during the first IVF cycle. When the same analysis was performed in patients receiving a micronutrient supplementation in the second IVF cycle, starting from three months before controlled ovarian stimulation, we observed an increased number of proteins with free thiol groups (Fig. 2b and d). Therefore, a significant recovery of a physiological level in the overall oxidative stress status in these biological fluids after treatment is demonstrated. Indeed, both in blood serum and follicular fluid of treated women, 2D electrophoresis detected numerous spots distributed between pH 4 and 7, with a wide molecular weight ranging from about 60 to 200 kDa (Fig. 2b and d). The quantitative analysis of main spots enabled us to highlight a statistically significant increase in spot intensity in both serum and follicular fluid recovered from women treated with micronutrient supplementation (p < 0.05) in comparison to untreated (Fig. 2). As regard to IVF outcome parameters, we observed that the mean number of retrieved oocytes did not differ between the two cycles, whereas the mean number of oocytes not suitable for injection was significantly reduced (1.20 ± 0.77 vs. 1.88 ± 1.01; P = 0.01) in the micronutrients supplemented cycles. Fertilization and cleavage rates as well as the mean number of top-quality transferred embryos did not differ significantly (Table 2). Nevertheless, in the treated group, we registered a total of 3 ongoing pregnancies (pregnancy rate =17.7 %), which represent an encouraging results in aged women undergoing to IVF.Fig. 1


Antioxidants reduce oxidative stress in follicular fluid of aged women undergoing IVF
Avidin-blotting after two dimensional electrophoresis of biotin-labeled free-SH residues in serum (a, b) and follicular fluid (c, d) from untreated patient (a, c) or patient treated with micronutrients supplementation (b, d). The values are the mean ± SD. Statistically significant difference in relative intensity is indicated (*P < 0.05)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5015196&req=5

Fig2: Avidin-blotting after two dimensional electrophoresis of biotin-labeled free-SH residues in serum (a, b) and follicular fluid (c, d) from untreated patient (a, c) or patient treated with micronutrients supplementation (b, d). The values are the mean ± SD. Statistically significant difference in relative intensity is indicated (*P < 0.05)
Mentions: Demographic features and clinical data of patients are presented in Table 1. Total FSH-LH units, number of days of stimulation, peak E2 levels at hCG administration were not significantly different between the two IVF cycles (without or with micronutrients supplementation) in the enrolled patients. In order to detect the oxidative stress levels, we measured the total antioxidant capacity and ROS induced-damage on proteins both in follicular fluid and in serum. By means of the FRAP assay we measured total antioxidant capacity showing a significant increase in TAC both in follicular fluid and in serum of women treated with micronutrient supplementation (Fig. 1). In order to confirm these data, we analyzed all samples by two dimensional electrophoresis to monitor the presence of free-SH residues in serum and follicular fluid proteins. In Fig. 2, a representative 2D-electroforesis of serum and follicular fluid proteins is showed. By this procedure, numerous spots distributed in a pH range of about 4–7 with an apparent molecular weight ranging between 15 and 75 kDa (Fig. 2a and c) were evident in patients not receiving micronutrient supplementation (untreated) during the first IVF cycle. When the same analysis was performed in patients receiving a micronutrient supplementation in the second IVF cycle, starting from three months before controlled ovarian stimulation, we observed an increased number of proteins with free thiol groups (Fig. 2b and d). Therefore, a significant recovery of a physiological level in the overall oxidative stress status in these biological fluids after treatment is demonstrated. Indeed, both in blood serum and follicular fluid of treated women, 2D electrophoresis detected numerous spots distributed between pH 4 and 7, with a wide molecular weight ranging from about 60 to 200 kDa (Fig. 2b and d). The quantitative analysis of main spots enabled us to highlight a statistically significant increase in spot intensity in both serum and follicular fluid recovered from women treated with micronutrient supplementation (p < 0.05) in comparison to untreated (Fig. 2). As regard to IVF outcome parameters, we observed that the mean number of retrieved oocytes did not differ between the two cycles, whereas the mean number of oocytes not suitable for injection was significantly reduced (1.20 ± 0.77 vs. 1.88 ± 1.01; P = 0.01) in the micronutrients supplemented cycles. Fertilization and cleavage rates as well as the mean number of top-quality transferred embryos did not differ significantly (Table 2). Nevertheless, in the treated group, we registered a total of 3 ongoing pregnancies (pregnancy rate =17.7 %), which represent an encouraging results in aged women undergoing to IVF.Fig. 1

View Article: PubMed Central - PubMed

ABSTRACT

Background: The status characterized by the imbalance between pro-oxidants and antioxidants molecules, defined as oxidative stress, has been suggested to be involved in the pathogenesis of subfertility in females. This study aims to evaluate the impact of a complete micronutrients supplementation on oxidative stress levels in follicular microenvironment as well as on in vitro fertilization (IVF) outcome.

Methods: This preliminary study was conducted between January 2014 and July 2015 at the Siena University Hospital Infertility Clinic. Serum and follicular fluid were collected from infertile women aged&thinsp;&gt;&thinsp;39&nbsp;years who underwent two in vitro fertilization cycles: in the first cycle they were treated with GnRH-antagonist protocol and gonadotropins for controlled ovarian hyperstimulation, whereas in the second cycle ovarian stimulation protocol was associated to micronutrients supplementation, starting three months earlier. Protein oxidation levels and total antioxidant capacity in serum and in follicular fluid were evaluated in IVF cycles with or without micronutrients supplementation. Differences in IVF outcome parameters were statistically evaluated.

Results: Two-dimensional electrophoresis analyses demonstrated that when patients assumed micronutrients before IVF cycles, follicular fluid and serum proteins were protected from oxidative damage. Comparable results were obtained when total antioxidant capacity was measured. Moreover, the mean number of good quality oocytes retrieved when patients received micronutrients supplementation was significantly increased.

Conclusion: The additional treatment with micronutrients, starting three months before IVF cycles, protects the follicular microenvironment from oxidative stress, thus increasing the number of good quality oocytes recovered at the pick up.

No MeSH data available.